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Development of de novo donor-specific anti-HLA antibody (dnDSA) is associated with poor graft survival in adults. However, there is a paucity of data about its prevalence and outcome in Indian children. We retrospectively assessed the proportion and spectrum of dnDSA and its outcome on antibody-mediated rejection (ABMR) and graft function. Children ≤18 years who were transplanted between November 2016 and October 2019 were included in this study. Pretransplant donor-specific antibody (DSA) was screened by complement-dependent cytotoxicity, flow cytometry crossmatch, and single antigen bead (SAB) class I and II by Luminex platform. Either antithymocyte globulin or basiliximab was used as induction. Tacrolimus, mycophenolate, and prednisolone were used for the maintenance of immunosuppression. SAB screening was done at 1, 3, 6 months, and yearly in seven children and at the time of acute graft dysfunction in eight. Mean fluorescence intensity ≥1000 was considered positive. Protocol biopsies were done at 3, 6, and 12 months and annually thereafter in seven children. Fifteen children, all males with a median age (interquartile range) of 13 years (11; 15.5) were analyzed. Only one child had pretransplant DSA who developed dnDSA posttransplant. Overall, 8 (53%) developed dnDSA over a median follow-up of 18 months. Seven (87%) had Class II, one Class I and 3 (37%) both Class I and II. Six had dQ and two had DR. All children with dnDSA had ABMR, of these two had subclinical rejection. DSAs persisted despite treatment, though graft function improved. Children with DSA and ABMR had lower graft function than those without DSA. The proportion of dnDSA was high in our study, majority against DQ. The detection of dnDSA prompted early diagnosis and treatment of ABMR., (Copyright © 2023 Copyright: © 2023 Saudi Journal of Kidney Diseases and Transplantation.)

Background: Berberine is a plant-derived alkaloid with potent anti-cancer activities. Berberine may redirect the tumor-promoting immunosuppressive M2 macrophages, to tumoricidal activated M1 macrophages. But such an anti-tumor function remains to be demonstrated., Hypothesis: Polarization of macrophages to an immunosuppressive phenotype within the tumor microenvironment promotes tumor growth and contributes to resistance to chemotherapy. We examined if berberine would target macrophage polarization to reinstate anti-tumor immune response., Study Design: Using a B16F10 mouse melanoma model, we assessed berberine-induced re-polarization of immunosuppressive M2 macrophages to anti-tumor M1 macrophages and subsequent T-cell activation within the immunosuppressive tumor microenvironment., Methods: The B16F10 culture supernatant along with tumor antigen was used as tumor mimicking conditioned medium (CM). The bone marrow-derived macrophages were cultured in CM for 5 days. The CM-induced skewing of macrophages to M2-like phenotype was confirmed by flow cytometry and ELISA. The T-cells were co-cultured with macrophages to decipher the effect of berberine on T-cell differentiation. In vivo efficacy of berberine was analyzed using melanoma model of solid tumor., Results: Berberine inhibited rIL-6-induced STAT-3 phosphorylation and IL-10 release from B16F10 cells. It enhanced tumor antigen-induced IL-1β, IL-12 and TNFα, but suppressed IL-6 and TGF-β release. Berberine significantly prevented the tumor antigen-mediated IL-10-enhanced IL-6 and TGF-β expression. The CM skewed the bone marrow-derived macrophages to CD206-high but MHC-II-low M2-like tumor-associated macrophages. Berberine partially prevented the generation of these macrophages and was associated with reduced C/EBPβ and Egr2 mRNA expression and lowered IL-10 and TGF-β production. Berberine significantly reduced Arginase-1 expression in CM-treated M1 and M2-like macrophages. Berberine increased MHC-II and CD40 expression on the macrophages augmenting the CTL activity and the number of IFNγ-producing CD4 + T-cells. Berberine significantly lowered tumor volume, weight and enhanced the frequency of M1-like macrophages in mice., Conclusion: These data indicate that berberine interferes with pro-tumor macrophage polarization and IL-10 and TGF-β release but restores Tcell anti-tumor cytotoxicity in the tumor microenvironment., (Copyright © 2021. Published by Elsevier GmbH.)

In the present study, the statistical forensic parameters were evaluated for the loci present in PowerPlex 21 autosomal and PowerPlex 23 Y-STR multiplex systems in 168 unrelated individuals living in the state of Uttar Pradesh, India. The combined discrimination power (CPD) and combined exclusion power (CPE) was 1 and 0.999999 respectively for all 20 autosomal STR loci. Penta E showed the greatest (0.980) and CSF1PO showed the lowest (0.855) power of discrimination in the studied population. The haplotype diversity for 23 Y-STR loci was observed to be 0.999. The study also presents the first global report on polymorphism on D1S1656, D6S1043 and D12S391 autosomal STR loci in the Indian population. The resulting data revealed that these STR multiplex systems are highly polymorphic and can be used for forensic purposes.

Background: The simultaneous, quantitative determination of all active ingredients present in the analgesic formulation (Dazzle ointment) requires an ideal and novel method by which these phytoconstituents can be separated with the highest resolution without any interference from one another. Objective: The present work was conducted to develop and validate a quantitative method for the simultaneous estimation of all five phytoconstituents present in a polyherbal analgesic ointment by GC. Methods: α-Pinene, 1,8-cineole, camphor, menthol, and methyl salicylate present in the ingredients of the ointment were analyzed and quantified by GC using a crosslinked 5% phenyl polydimethylsiloxane capillary column, nitrogen as a carrier gas, and a flame-ionization detector. Aniline was used as the internal standard. Method validation was also performed in order to demonstrate its selectivity, linearity, accuracy, precision, LOD, LOQ, and robustness. Results: The calibration curves of all five marker compounds showed good linear correlation coefficients (r² >0.998) within the tested ranges. The precision of the method was tested by carrying out intra- and interday analyses of the same sample. RSD values were observed to be <1.00%. The accuracy of the method, determined by performing recovery studies, was found to be between 99.25 and 101.39%. The developed method was also demonstrated to be robust (RSD <1.29%) by making small but deliberate variations in method parameters. Conclusions: The developed GC method is simple, precise, and accurate, it and can be used for the rapid quality control testing of the polyherbal formulation. Highlights: The developed GC method will assist in the standardization of polyherbal analgesic formulation consists of α-pinene, 1,8-cineole, camphor, menthol, and methyl salicylate as active constituents.

Background: To ascertain the validity of kidney paired donations (KPDs) as an alternative strategy for increasing living donor kidney transplantations (LDKTs) in an LDKT-dominated transplant programme since directed kidney transplantation, ABO-incompatible or crossmatch-positive pairs are not feasible due to costs and infectious complications., Methods: This was a prospective single-centre study of 77 KPD transplantations (25 two-way, 7 three-way and 1 six-way exchange) from 1 January 2015 to 1 January 2016 of 158 registered donor recipient pairs. During this period, a total of 380 kidney transplantations [71 deceased donor kidney transplantations (DDKTs), 309 LDKTs] were performed. The reasons for opting for KPD were ABO incompatibility ( n  = 45), sensitization ( n  = 26) and better matching ( n  = 6)., Results: KPD matching was facilitated in 62% ( n  = 98) of transplants. In all, 48.7% ( n  = 77) of the transplants were completed in 2015, whereas 13.3% ( n  = 21) of the matched patients were to undergo transplant surgery in early 2016 after getting legal permission. The waiting time for KPD was shorter compared with DDKT. The death-censored graft survival and patient survival were 98.7% ( n  = 76) and 93.5% ( n  = 72), respectively. In all, 14.2% ( n  = 11) of patients had acute rejection. Match rates among sensitized ( n  = 60) and O group patients ( n  = 62) were 58.3% ( n  = 35) and 41.9% ( n  = 26), respectively. Of these, 43.3% ( n  = 26) and 29% ( n  = 18) of transplants were completed and 15% ( n  = 9) and 12.9% ( n  = 8), respectively, are waiting for legal permission., Conclusions: LDKT increased by 25% in 1 year in our single-centre KPD programme. Our key to success was the formation of a KPD registry, awareness and active counselling programs and developing a dedicated team., (© The Author 2017. Published by Oxford University Press on behalf of ERA-EDTA.)

In a living donor kidney transplantation (LDKT) dominated transplant programme, kidney paired donation (KPD) may be a cost-effective and valid alternative strategy to increase LDKT in countries with limited resources where deceased donation kidney transplantation (DDKT) is in the initial stages. Here, we report our experience of 300 single-centre KPD transplantations to increase LDKT in India. Between January 2000 and July 2016, 3616 LDKT and 561 DDKT were performed at our transplantation centre, 300 (8.3%) using KPD. The reasons for joining KPD among transplanted patients were ABO incompatibility (n = 222), positive cross-match (n = 59) and better matching (n = 19). A total of 124 two-way (n = 248), 14 three-way (n = 42), one four-way (n = 4) and one six-way exchange (n = 6) yielded 300 KPD transplants. Death-censored graft and patient survival were 96% (n = 288) and 83.3% (n = 250), respectively. The mean serum creatinine was 1.3 mg/dl at a follow-up of 3 ± 3 years. We credit the success of our KPD programme to maintaining a registry of incompatible pairs, counselling on KPD, a high-volume LDKT programme and teamwork. KPD is legal, cost effective and rapidly growing for facilitating LDKT with incompatible donors. This study provides large-scale evidence for the expansion of single-centre LDKT via KPD when national programmes do not exist., (© 2017 Steunstichting ESOT.)

One third of healthy willing living kidney donors are rejected due to ABO blood group incompatibility and donor specific antibody. This increases pre-transplant dialysis duration leading to increased morbidity and mortality on the kidney transplantation waiting list. Over the last decade kidney paired donation is most rapidly increased source of living kidney donors. In a kidney transplantation program dominated by living donor kidney transplantation, kidney paired donation is a legal and valid alternative strategy to increase living donor kidney transplantation. This is more useful in countries with limited resources where ABO incompatible kidney transplantation or desensitization protocol is not feasible because of costs/infectious complications and deceased donor kidney transplantation is in initial stages. The matching allocation, ABO blood type imbalance, reciprocity, simultaneity, geography were the limitation for the expansion of kidney paired donation. Here we describe different successful ways to increase living donor kidney transplantation through kidney paired donation. Compatible pairs, domino chain, combination of kidney paired donation with desensitization or ABO incompatible transplantation, international kidney paired donation, non-simultaneous, extended, altruistic donor chain and list exchange are different ways to expand the donor pool. In absence of national kidney paired donation program, a dedicated kidney paired donation team will increase access to living donor kidney transplantation in individual centres with team work. Use of social networking sites to expand donor pool, HLA based national kidney paired donation program will increase quality and quantity of kidney paired donation transplantation. Transplant centres should remove the barriers to a broader implementation of multicentre, national kidney paired donation program to further optimize potential of kidney paired donation to increase transplantation of O group and sensitized patients. This review assists in the development of similar programs in other developing countries., Competing Interests: Conflict-of-interest statement: All the authors have no conflicts of interests to declare.

Aim: To report the first international living related two way kidney paired donation (KPD) transplantation from India which occurred on 17 th February 2015 after legal permission from authorization committee., Methods: Donor recipient pairs were from Portugal and India who were highly sensitized and ABO incompatible with their spouse respectively. The two donor recipient pairs had negative lymphocyte cross-matching, flow cross-match and donor specific antibody in two way kidney exchange with the intended KPD donor. Local KPD options were fully explored for Indian patient prior to embarking on international KPD., Results: Both pairs underwent simultaneous uneventful kidney transplant surgeries and creatinine was 1 mg/dL on tacrolimus based immunosuppression at 11 mo follow up. The uniqueness of these transplantations was that they are first international KPD transplantations in our center., Conclusion: International KPD will increases quality and quantity of living donor kidney transplantation. This could be an important step to solving the kidney shortage with additional benefit of reduced costs, improved quality and increased access for difficult to match incompatible pairs like O blood group patient with non-O donor and sensitized patient. To the best of our knowledge this is first international KPD transplantation from India., Competing Interests: Conflict-of-interest statement: There are no conflicts of interest to report.

Aim: To estimate population parameters based on allele frequencies obtained for 15 polymorphic autosomal STR loci investigated in caste and tribal populations of central India (n = 419)., Methods: Multiplexed PCR amplifications of the 15 Autosomal STR Loci were performed and amplified products were genotyped using multi-capillary electrophoresis on an ABI 3100 genetic analyser. Parameters of population genetics and forensic interest based on the allele frequencies were calculated. Genetic affinity of the studied populations among themselves and with previously reported populations of India was also analysed using distance-based NJ tree and using PCA plot., Results: All the 15 STR loci were highly informative and discriminating, with CPD of 0.999 99. Except for Brahmins and Rajput, all other studied populations were in Hardy-Weinberg equilibrium (HWE). The only tribe (Gond) population studied showed significant variation with the other four caste populations (Brahmin, Yadav, Rajput and Muslim) studied and formed a cluster with other previously reported tribal populations of India. Nei's genetic distance based clustering pattern of the NJ tree and the PCA plot showed the same pattern of genetic relationship, i.e. caste and tribal populations formed a distinct cluster., Conclusions: With respect to the distribution of alleles at each STR locus, the studied loci were found to be substantially polymorphic in all the studied populations, indicating good informativeness of all 15 STR markers. The population data generated in this study are useful for forensic, anthropological and demographic studies.

The aim of this study was to contribute new data on autosomal STR and Y-STR markers of the Majhi tribal community of Chhattisgarh, a state of central India. In order to improve available databases of forensic interest, we analyzed 15 autosomal STR markers in a population sample of 129 unrelated indigenous Majhi tribe and 23 Y-chromosomal STR markers in the 107 males of the sample. The combined power of discrimination (CPD) and combined power of exclusion (CPE) were greater than 0.999999 and 0.999998 respectively for autosomal STRs. In addition, a total of 64 distinct Y-STR haplotypes were observed out of which 31 Y-STR haplotypes were observed only once. The haplotypes diversity and discrimination capacity for 23 Y-STR loci was 0.989 and 0.589, respectively. The highest gene diversity values at the single copy locus DYS570 and the multi-copy locus DYS385 a/b were 0.805 and 0.952, respectively. Average gene diversity over loci was 0.652304 ± 0.325572.

Aim: To avoid desensitization protocols and ABO incompatible kidney transplantation (KT) due to high costs and increased risk of infections from intense immunosuppression., Methods: We present institutional ethical review board - approved study of single center 6-way kidney exchange transplantation. The participants comprised ABO incompatibility ( n = 1); positive cross-match and/or presence of donor specific antibody ( n = 5). The average time required from registration in kidney paired donation (KPD) registry to find suitable donors was 45 d and time required to perform transplants after legal permission was 2 mo., Results: Graft and patient survival were 100%, and 100%, respectively. One patient had biopsy-proven acute borderline T cell rejection (Banff update 2013, type 3). Mean serum creatinine was 0.8 mg/dL at 9 mo follow-up. The waiting time in KPD was short as compared to deceased donor KT., Conclusion: We report first non-simultaneous, single center, 6-way kidney exchange transplantation from India. Our experience will encourage other centers in India to undertake this practice., Competing Interests: Conflict-of-interest statement: There are no conflicts of interest to report.

An analysis of 15 autosomal short tandem repeat (STR) loci and 17 Y-STR loci was performed in 123 unrelated members of the Oraon tribal community of Central India. The combined power of discrimination (CPD) and combined power of exclusion (CPE) were greater than 0.99999 and 0.999989, respectively, for autosomal STRs. In addition, a total of 58 distinct Y-STR haplotypes were observed out of which 54 Y-STR haplotypes were observed only once. The haplotype diversity and discrimination capacity for 17 Y-STR loci was 0.997 and 0.906, respectively.

Aims: Seventeen Y-STR loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385a/b, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635 and Y-GATA-H4) were analysed in 173 males belonging to the central Indian population with the aim of studying genetic diversity and adding to the population database., Methods: Multiplexed PCR amplifications of the 17 Y STR loci were performed using AmpFlSTR® Yfiler® Kit. Amplified products were genotyped using a multi capillary electrophoresis with POP-4 polymer in ABI Prism 3100 Genetic Analyzer. Population genetic diversity and allele frequencies were calculated. The haplotype data obtained in the study was compared with the Y-STR haplotypes reference database (YHRD, http://www.yhrd.org ) and with previously published population data using the AMOVA tool and visualised in two-dimensional multidimensional scaling (MDS) plots., Results: A total of 147 haplotypes were observed, out of which 125 were unique. Haplotype diversity and discriminating capacity were found to be 0.9979 and 0.8497, respectively. The gene diversity at the loci ranged from 0.398-0.785. Genotype diversity at the locus DYS385a/b was found to be 0.869., Conclusions: The population of central India was found to be significantly different (p < 0.05) when compared with populations from other parts of the Indian sub-continent and the population data of other countries. The population data generated in this study are useful for forensic, anthropological and demographic studies.

Aims: Genotypic polymorphism studies at 15 highly polymorphic short tandem repeat (STR) loci were carried out in Bhil tribal population of Madhya Pradesh, in central region of India., Methods: The analysis of 15 autosomal STR loci (TH01, D3S1358, vWA, D21S11, TPOX, D7S820, D19S433, D5S818, D2S1338, D16S539, CSF1PO, D13S317, FGA, D18S51, D8S1179) was done in 183 unrelated individuals of the Bhil tribe., Results: Heterozygosity among the studied 15 autosomal STR loci ranged from 63.5-86.5%. The loci D2S1338 and FGA were found to be mostly polymorphic. All loci fall under Hardy-Weinberg equilibrium, except loci D18S51 and D7S820. These STR loci were highly informative and discriminating, with CPD > 0.99999., Conclusion: The clustering pattern of the NJ tree and the PCA plot showed grouping of the Bhil population with previously reported tribes of central India.

The analysis of 15 autosomal STR locus (TH01, D3S1358, vWA, D21S11, TPOX, D7S820, D19S433, D5S818, D2S1338, D16S539, CSF1PO, D13S317, FGA, D18S51, D8S1179) was done in 582 healthy unrelated individuals (Male-366, Female-216) originating from the various geographical regions of Madhya Pradesh, India. All locus fall under Hardy-Weinberg equilibrium except TPOX. These STR loci were highly informative and discriminating with combined power of discrimination (CPD) >0.99999. Locus wise allele frequencies of the studied population were compared with the other published populations. Also the Clustering pattern and genetic distance of studied populations is compared and presented with various populations. The studied population showed the genetic proximity with geographically close populations of India and significant genetic variation with distant populations which is also evident by clustering pattern of the NJ tree and the PCA plot.

The analysis of 12 X STR loci (DXS10103, DXS8378, DXS7132, DXS10134, DXS10074, DXS10101, DXS10135, DXS7423, DXS10146, DXS10079, HPRTB and DXS10148) belonging to four linkage group was done in 183 (100 males and 83 females) unrelated members of Bhil population. Heterozygosity among the studied 12 X STR loci showed a distribution of from 59.7% to 92.8%. No significant difference was recorded in the allele frequencies of males and females. The loci DXS10135 and DXS10101 were found to be most polymorphic. Haplotype diversity was found to be higher than 0.990 for all the four linkage groups. A total of 86, 69, 71 and 71 haplotypes were observed for linkage group I, II, III and IV, respectively. The results showed departure from Hardy-Weinberg equilibrium with respect to three loci DXS10079, DXS10135 and DXS10101. This is first report on these 12 X STR markers from India. All the loci in the Argus X 12 kit were fairly informative in the Bhil population and the population showed significant genetic variation with all the compared populations from other parts of the world., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

The presence of positive anti-human, globulin-enhanced, complement-dependent cytotoxicity crossmatches (AHG-CDC-XM) generally has been considered as a contraindication to kidney transplantation (KTx). Flow cytometry crossmatch (FCXM) and solid phase antibody screening are most sensitive and specific methods for immunological assessment. The outcome effect of donor-specific antibody (DSA) positive by Luminexx platform and CDCXM negative has been evaluated, but not the outcome of CDCXM positive and DSA negative. This is a case report describing KTx in a patient with pre-transplant positive AHG-CDC-XM but negative FCXM and without detectable DSA by Luminexx single antigen beads. CDCXM, FCXM, DSA were negative after transplantation, and kidney allograft biopsy did not show immune injury with negative C4d. Our report suggests that KTx with positive AHG-CDC-XM can be considered if FCXM and DSA were negative with careful immunological monitoring after transplantation. In our case, the IgM-antibodies were responsible for the positive AHG-CDC-XM result. The patient with positive CDC XM has a good outcome in the absence of DSA. Further work is needed to determine under what circumstances CDCXM positive transplants can be performed with FCXM and DSA negative.

Recurrence of primary focal segmental glomerulosclerosis (FSGS) is an important cause of graft loss after renal transplantation (RTx). We report our experience in 34 patients with primary FSGS who underwent RTx between April 1999 and June 2009, using hematopoietic stem cell transplantation (HSCT). They belonged to four groups: group 1 (n = 12) received high-dose HSCT in periphery, thymus, bone-marrow, and portal circulation with low-dose non-myeloablative conditioning; group 2 (n = 7) was modified with HSCT without marrow/thymic infusion; and group 3 (n = 3) received HSCT and proteasome inhibitor Bortezomib replacing conditioning. Group 4 (n = 12), were controls who opted for RTx under standard triple-drug immunosuppression. Patient/donor demographics were comparable in all. No recurrence was noted in group 1 with mean follow-up of 8.1 years, whereas 28.6% of group 2, 33.3% of group 3, and 36.4% of group 4 had recurrence over mean follow-up of 2.6, 1.1, and 6.5 years, respectively. Mean serum creatinine was 1.62, 1.69, 1.41, and 1.73 mg%, respectively. Rejections were noted in 41.7%, 28.6%, 0%, and 45.5% grafts, respectively. Groups 1 and 4 had 25% patient loss each, group 2 had 28.6% loss, and no loss was observed in group 3. Graft loss was noted in 33.3% in group 1, 14.3% in group 2, nil in group 3, and 16.7% in the last group. Recurrent FSGS was prevented in RTx with HSCT in thymic, marrow infusion under low-dose non-myeloablative conditioning compared to controls and Bortezomib group, thus suggesting potential role of central tolerance in FSGS.

Background/objective: We designed a clinical trial on a group of live-donor renal transplantation (LDRT) patients subjected to pre-transplant stem cell transplantation (SCT) to minimize immunosuppression to low-dose steroid monotherapy., Methods: LDRT patients subjected to pretransplant SCT who had stable graft function for ≥2 years and serum creatinine (SCr) <2 mg/dL were recruited. Patients with diabetes, hepatitis C/B, rejections, or unwilling to participate, were excluded. They had been subjected to non-myeloablative conditioning of total lymphoid irradiation (TLI)/bortezomib and cyclophosphamide, rabbit-antithymocyte globulin (r-ATG) and rituximab with SCT. The maintenance immunosuppression consisted of calcineurin inhibitors (CNI) and/or anti-proliferative agents and prednisone. Donor-specific antibodies (DSA) and peripheral T-regulatory cells (CD127(low/-)/4(+)/25(high)) (p-Tregs) were studied before and after withdrawal of major immunosuppressants; graft biopsy was taken after 100 days of withdrawal in willing patients. Rejections were planned to be treated by anti-rejection therapy followed by rescue immunosuppression., Results: All immunosuppression but prednisone, 5-10 mg/day has been successfully withdrawn for a mean of 2.2 years in 76 patients with a mean age of 31.4 years and a mean donor-recipient HLA match of 2.9. The mean SCr of 1.4 mg/dL and p-Tregs of 3.5% was remained stable after withdrawal; DSA status was negative in 35.5% and positive in 47.4% patients. Protocol biopsies in all 10 patients who gave the consent were unremarkable., Conclusion: Stable graft function in LDRT on low-dose steroid monotherapy using pre-transplant SCT under non-myeloablative conditioning with generation of p-Tregs can be achieved successfully and safely.

Objectives: Cytomegalovirus (CMV) is a common opportunistic infection following renal transplantation (RTx). It responds promptly to antiviral treatment. The mortality rate reaches 90% if untreated. Identification of risk factors helps in the early diagnosis of CMV. We studied demographic features, risk factors, and outcomes associated with CMV infection in RTx recipients despite ganciclovir prophylaxis., Materials and Methods: We reviewed 720 RTx recipients between 2007 and 2009. We examined the serostatus of the donor and recipient before transplantation using an enzyme-linked immunosorbent assay, and diagnosed CMV infections in recipients by CMV DNA detection with a polymerase chain reaction., Results: A total of 42 of 750 (5.6%) patients were identified to display CMV infection (69.1%) or disease (30.9%). Their mean age was 34 ± 13.5 years, with 80.9% men. CMV serologic status was D+/R- in 21.4% and D+/R+ in 59.5% patients. Fever, malaise (76.2%), and leukopenia (52.3%) were the commonest presenting symptoms; diabetes (30.9%) and hepatitis C virus (28.6%) the commonest comorbid conditions. Risk factors were triple drug immunosuppression (47.6%), antithymocyte globulin ATG induction (54.8%), and a rejection episode (26.1%) and methylprednisolone (76.2%) which were more common in CMV disease than infection. Mean CMV DNA at diagnosis was 78,803; 71.2% patients developed CMV within 6 months posttransplantation, the majority occurring after 3 months. With a mean follow-up of 4 ± 1.9 years, patient and graft survival rates were 85.7% and 81% with a mean serum creatinine value of 1.83 ± 12 mg/dL., Conclusions: Universal CMV prophylaxis was associated with a low incidence (5.6%) and mild form of CMV disease among our patients., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Highly sensitized patients are destined to remain untransplanted for long. Early transplantation results in cost-saving, reduced morbidity/mortality and improved quality of life. We carried out a prospective study to evaluate the efficacy and safety of desensitization protocol vis-à-vis patient/graft survival in living donor renal transplantation in highly sensitized patients. Between December 2008 and April 2010, 34 renal transplant (RTx) patients underwent desensitization protocol. An anti-human globulin-enhanced lymphocytotoxicity crossmatch assay (AHG-CDC) ≥25% and T-cell median channel shift (MCS) >50, B-cell MCS >100 [flow crossmatch (FXM)] were considered crossmatch (XM) positive. All patients were administered bortezomib (1.3 mg/m 2 , days 1, 4, 8, 11), plasmapheresis, rabbit-anti-thymocyte globulin (r-ATG), mycophenolate mofetil (MMF) and intravenous immunoglobulins (IVIg). LCXM and FXM were repeated post-protocol. In the event of persistent sensitization, additional bortezomib cycle was repeated along with plasmapheresis, IVIg, calcineurin inhibitors (CNI) and rituximab. If the cross match (CMX) was negative or acceptable, patients underwent RTx. Post-transplant immunosuppression consisted of prednisone, CNI and MMF. Biopsy was performed in the event of graft dysfunction and treated accordingly. There were 18 males and 16 females, with a mean age of 37.4 years. Mean dialysis duration was 14.9 ± 17.6 months. Average third party transfusions were 6.2 ± 4.5, 17.6% had autoimmune diseases, 20.6% were multi-para. Pre-protocol AHGXM was 55.3 ± 24.5%, T-cell crossmatch (TCXM) was 122.4 ± 91.4 MCS and B-cell crossmatch (BCXM) was 279 ± 142.9 MCS. Totally, 85.3% responded within 1 month with reduction in AHG-CDC to 19.9 ± 5.2%, TCXM to 24.7 ± 19.4 MCS and BCXM to 74.7 ± 34.8 MCS. Side effects noted in 38.2% were manageable. Over follow-up of 0.92 ± 0.8 years, patient/graft survival was 100%/88.2% and mean serum creatinine was 1.27 ± 0.32 mg/dL. Acute rejections were noted in 24.1%, who responded to steroids + rabbit antithymocyte globulin (rATG). Five (14.7%) patients were transplanted after changing donors. Our desensitization protocol seems to be safe and effective. Bortezomib may offer new possibilities in desensitization protocols.

Background: Current treatments for autoantibody-mediated diseases (i.e., systemic lupus erythematosus) and alloantibodies (in transplant) are minimally effective. Although they deplete naïve B cells, plasmablasts, and transiently reduce antibody concentrations, they are minimally effective against long-lived, antibody-producing plasma cells. In transplantation, plasma cells produce antibodies directed against human leukocyte antigen (HLA) antigens causing poor allograft survival. We report the first clinical experience with a plasma cell depleting therapy, bortezomib, to abrogate anti-HLA antibodies in transplantation (outside of rejection) in an attempt to improve long-term allograft survival., Methods: Eleven patients with anti-HLA alloantibodies were treated with bortezomib. All patients underwent plasmapheresis to aid in removal of antibodies and to determine the effect of bortezomib. Serial measurements of anti-HLA antibody levels were conducted weekly by single antigen bead on Luminex platform., Results: Bortezomib treatment elicited substantial reduction in both donor-specific antibody (DSA) and non-DSA levels. Antibodies were directed against DSA in 8 of 11 cases. Mean time to antibody appearance was 2 months posttransplant. Within 22 days (median) from treatment initiation, 9 of 11 patients' antibody levels dropped to less than 1000 mean fluorescence intensity. Of two patients without successful depletion, all had peak mean fluorescence intensity more than 10,000. At a mean follow-up of approximately 4 months posttreatment, all patients have stable graft function. Minimal transient side effects were noticed with bortezomib in the form of gastrointestinal toxicity, thrombocytopenia, and paresthesias., Conclusions: Bortezomib therapy effectively abrogates anti-HLA antibodies. Hence, removal of antibodies, by proteasome inhibition, represents a new treatment strategy for transplantation and may have benefit in autoimmune-related disease.

A total of 69 individuals received a kidney from a living donor after a TLI-based clonal deletion protocol with no post-transplant maintenance immunosuppression planned. If needed, immunosuppression was started on a patient-specific basis, adding one drug at a time, a strategy we AWN". call "Drugs Added When Needed," or "DAWN. Following this strategy, at last follow-up 40 of the 69 patients (58%) had to be rescued by conventional immunosuppression, 23 (33%) had to be started on daily prednisone and six (9%) remained with no maintenance immunosuppression. The overall rate of de novo donor-specific antibody produced was 36% (in 25 of the 69 patients), and mean time to detection was about four months. The incidence of acute rejection episodes that displayed humoral components was 27% (19 cases), of which 14 were pure antibody-mediated rejection, five combined antibody- and T-cell-mediated rejection, and six were episodes (9%) of pure T-cell-mediated rejection. Finally, this study shows that although complete clonal deletion was not achieved, an important proportion of patients--42%, or 29 of the original 69--could be maintained with prednisone alone or even with no immunosuppression for a total mean follow-up of 13.3 months. Moreover, 16 patients with recent follow-up are surviving with no maintenance immunosuppression or just on prednisone. The mean serum creatinine at last follow-up for these 16 patients is 1.33 +/- 0.2 mg/dL with a mean follow-up of 19.3 months. Clonal deletion can be used to transplant patients without maintenance immunosuppression, adding drugs only as needed.

We show the ability of bortezomib to remove donor-specific HLA antibody from kidney allograft patients, the drug acting as a proteasome inhibitor, providing targeted therapy against antibody-producing plasma cells. Ten out of thirteen patients (77%) experienced primary DSA reversal, and in the remaining three patients the MFI of their primary DSA was dramatically reduced. Bortezomib is a viable therapy to treat donor-specific HLA antibody in allograft recipients. The potential for long-term benefits--and complications--are still unknown. Prospective trials are being conducted at the University of Cincinnati, Cincinnati, OH; at the Mayo Clinic, Rochester, MN; and at IKDRC-ITS, Ahmedabad, India.

1. A total of 61 patients were treated with a clonal deletion protocol and transplanted without planned post-transplant immunosuppression. 2. Twenty-nine (48%) patients did not develop any donor-specific anti-HLA antibodies after the transplant, with a median follow-up of 158 days and a mean sCr of 2.1 mg/dL at the last follow-up. 3. Only 23% of the patients who received a DST of 60 mL produced DSA after the transplant, while 68% of the patients who received a bigger DST dose did. 4. Small doses of donor-specific transfusions (60 mL) elicited smaller specific responses, allowing efficient deletion of the reacting clones, creating conditions in which donor-specific anti-HLA antibodies were not produced. 5. A better deleting agent is needed to achieve higher rates of success using the clonal deletion protocol.

Immunofluorescence (IF) studies are important diagnostic tool in understanding pathogenesis involved in graft injury. Acute humoral rejection (AHR) associated with circulating donor-specific cytotoxic antibodies, is a poor prognosticator for graft survival. It can be diagnosed by staining for C4d antibody using indirect IF technique. C4d staining required to diagnose AHR was made mandatory for reporting renal allograft biopsies in 7th Banff conference. We present 2 years experience of IF studies using C4d polyclonal antibody on 546 renal allograft biopsies belonging to two groups of patients; 464 from group A (tolerance induction protocol) and 82 from group B (controls). We observed C4d focal positivity in 4 (0.9%) biopsies from group A and 4 (4.9%) from group B. We conclude that it is advisable to collect simultaneous core biopsy samples for IF studies and light microscopy to give better definition of allograft injury and thereby support in clinical management.

We designed a prospective clinical trial of 357 patients divided in two groups--treated (n = 201) and controls (n = 156)--to evaluate effects of donor hematopoietic stem cell transplantation (HSCT) with minimal nonmyeloablative conditioning for tolerance induction in living related donor renal allograft recipients. Conditioning included donor leukocyte infusions, target-specific irradiation, anti-T-cell antibody, cyclophosphamide, cyclosporine (CsA), followed by bone marrow (BM)-derived and peripheral blood stem cell (PBSC) infusion into thymus, liver, BM, and periphery, with mean total dose of 20 x 10(8) nucleated cells/kg body weight (BW) (mean CD34(+) count: 0.9%) pretransplantation. CsA (3 mg/kg BW/d) and prednisolone (10 mg/d) were used for immunosuppression. Azathioprine/mycophenolate mofetil were added in the event of an acute rejection episode. The controls underwent transplantation with three drug immunosuppression. With a mean follow-up of 21.5 months, the treated cohort showed better allograft function with mean serum creatinine (SCr), 1.42 +/- 0.31 mg% in contrast with the controls mean SCr, 1.61 +/- 0.52 mg% (P < .0001) at 23.9 months follow-up. One-year allograft/patient survival was 95%/96.7% versus 89%/93.4%, respectively. Peripheral blood chimerism by fluorescent in situ hybridization was 0.8% +/- 0.2% in the subset of treated patients with gender-mismatched donors. No graft-versus-host disease was noted. Nine patients with donor-specific cytotoxic alloantibodies pretransplantation showed a decrease in positivity to <15% post-HSCT and were transplanted safely. A transient rise in donor-specific cytotoxic alloantibodies was noted in 19 treated patients post-HSCT, 14 of whom returned to the transplantable range within 2 weeks and five required a desensitization protocol. "Prope" tolerance may be induced in living related donor renal transplantation across major histocompatability complex barriers using HSCT with minimal nonmyeloablative conditioning.

In a developing country such as India, cadaveric renal transplantation accounts for only less than 1% of total renal transplantations. The reasons for such a low rate of cadaveric transplantation are many, ranging from lack of awareness to socioeconomic reasons. Our institute conducted a statewide public awareness program and initiated an intercity organ harvesting program. This doubled the cadaveric renal transplantations in the last 2 years. We performed 38 cadaveric transplantations among 190 renal transplantations in the last year (August 2005 to July 2006). We retrieved kidneys from 21 donors, of whom 9 were outside our city. From 21 donors we transplanted 38 recipients; out of whom 3 received dual kidneys and one kidney was discarded. The Mean age of the donors was 41.4 +/- 18.2 years with a mean cold ischemia time of 6.9 +/- 3.8 hours. Sixty-eight percent had delayed graft function. At the last follow-up, which was 190 +/- 98 days, patient survival rate was 90%: 4 patients died, including 2 due to bacterial sepsis and 2 due to cytomegalovirus (CMV) disease. The Graft survival rate was 85%, and the death-censored graft survival rate was 90%. Mean serum creatinine value at the last follow-up was 1.2 +/- 0.3 mg%. There were 5 episodes of acute rejection in 31 patients during first 3 months (16% acute rejection rate). The increase in cadaveric transplantations was associated with satisfactory patient and graft survival despite the high incidence of delayed graft function.

Human leukocyte antigens (HLA), the human version of the major histocompatibility complex (MHC), an integral part of maintenance of immune surveillance, have been widely studied for their roles in transplantation biology. A donor with an identical HLA system can donate tissue more successfully than the one who is not matched. The MHC is divided into class I, II, and III antigens; class I and II play important roles in transplantation immunology. HLA is codominantly expressed on chromosome 6 in every individual; HLA-A, -B, and -DR is known as the "haplo-type." There are two sets of HLA antigens in each individual. Thus a child can inherit four different haplo-type combinations from parents. There is a 25% chance of totally matched or mismatched siblings and a 50% chance of half-matched siblings among a family with parents being a 50% match. The main purpose of HLA typing and lymphocyte crossmatching (LCM) in transplantation is to assess donor-recipient immune compatibility and identify the presence of preformed donor-specific cytotoxic alloantibodies in the recipient. It can be tested by serology or molecular techniques. We studied 8462 individuals for HLA typing by serology supplemented with molecular techniques (sequence-specific primers with low resolution). The common alleles were HLA-A19 (9.4%), -A1 (7.7%), -A2 (7.2%), -B5 (10.2%), -B35 (6.6%), -B40 (5.3%), -DR2 (10.2%), -DR5 (7.5%), and -DR7 (5.1%). HLA typing and LCM testing support successful transplantation.

Introduction: Autoimmune disease represents a (AD) breakdown of natural tolerance against autoreactive antigens leading to a high mortality and morbidity. The reaction is usually polyclonal; T- and B-cell components of the hematopoietic system are responsible for disease progression. Allogeneic/autologous hematopoietic stem cell transplantation (HSCT) are the current modalities for treating drug-resistant AD., Patients and Methods: We present a single-center retrospective evaluation of allogeneic HSCT with nonmyeloablative, low-intensity conditioning in nine patients (five males, four females) with pemphigus vulgaris (PV) and 27 patients with systemic lupus erythematosus (SLE; 3 males, 24 females). The mean follow-up period was 4.24 years for PV and 4.9 years for SLE. Cytokine-mobilized HSC from unmatched related donors, with mean dose of 21.3 x 10(8) nucleated cells/kg body weight (BW; mean CD34(+) count, 6 x 10(6)/kg BW) was administered in to the thymus as well as the portal and peripheral circulations of recipients. Cyclosporine (4 +/- 1 mg/kg BW per day) and prednisolone (10 mg/kg BW per day) were administered for 6 months to protect mixed chimerism. A subset of patients with cross-gender donors were analyzed for peripheral blood chimerism at 1 month post-HSCT and every 3 months thereafter., Results: Sustained clinical remission with peripheral lymphohematopoietic chimerism of 0.7 +/- 0.3% was observed in PV, whereas SLE relapsed after mean of 7.35 months of disease-free interval associated with fall in chimerism from 5 +/- 3% to < or =0.08 +/- 0.03%., Conclusion: HSCT was effective to achieve early clinical remission of PV; and in SLE relapsed after a 7.35-month disease-free interval accompanied by a fall in mixed lymphohematopoietic chimerism.

Objective: We designed a prospective, randomized, and controlled clinical trial to evaluate the efficacy and safety of achieving a mixed chimerism-associated tolerance protocol for recipients of living related donor (LRD) renal allografts., Patients and Methods: Sixty-six consecutive patients were divided into two equal groups of 33 patients with end-stage renal disease. They were enrolled for transplantation after negative lymphocytotoxicity cross-matching (LCM). Both groups (treated [Tn] and control [Cn]) showed similar clinical and laboratory parameters and donor HLA match profiles. The Tn group underwent thymic transplantation of donor renal tissue, two donor-specific transfusions, low-intensity conditioning, and high-dose hematopoietic stem-cell transplantation (HSCT) before renal transplantation. The conditioning regimen included low-dose, target-specific irradiation (to abdominal and inguinal lymph nodes, bone marrow [BM] from thoracolumbar vertebrae and part of the pelvis on alternate days, 100 rad x 4), anti-T-cell antibodies (1.5 mg/kg body weight [BW]), cyclophosphamide (10 mg/kg BW x 2 consecutive days), and cyclosporine (CyA; >3 mg/kg BW/d). Unfractionated HSCT procured from the donor marrow was administered into the BM, portal and peripheral circulations, within 24 hours of achieving CD 4+/CD 8+ T-cell count less than 10% of normal. This infusion was supplemented with a dose of peripherally mobilized stem cells (mean total dose of 20 x 10(8) cells/kg recipient BW) administered peripherally. Renal transplantation was performed after negative LCM. Donor-specific cytotoxic antibodies were eliminated with intravenous immunoglobulins and plasmapheresis before renal transplantation. Mixed chimerism was evaluated before and after transplantation at monthly intervals in patients with donors of opposite gender by the FISH technique. Both groups received CyA and prednisolone for immunosuppression; Cn subjects also received mycophenolate mofetil/azathioprine. Rejection was treated with standard treatment. Immunosuppression was withdrawn 6 months after renal transplantation for patients with consistently positive chimerism. Clinical tolerance was defined as stable allograft function for more than 100 days without immunosuppression and confirmed by allograft biopsy., Results: Over a mean follow-up of 210 days, all Tn patients showed stable allograft function with mean serum creatinines (SCr) of 1.20 mg/dL, no rejection/CMV infections/graft or patient loss. A low-level donor-specific cytotoxic antibody was observed in all Tn patients. The CyA toxicity was noted in 10 (30.3%) patients. Persistent mixed hematopoietic chimerism was seen in all 21 patients irrespective of donor-recipient HLA matching (mean 0.5% before and 1 +/- 0.3% after transplantation). All four patients on drug withdrawal have shown donor-specific tolerance at a mean follow-up of 129.8 days. Other Tn patients are in the process of being weaned off immunosuppression. Mean SCr of controls was 1.45 mg/dL over a mean follow-up of 216 days. Acute rejection was observed in 17 (51.5%) patients; no CMV infection/patient loss was noted and one (3.03%) graft was lost in controls. No patient was lost in controls. No graft-versus-host disease was observed in Tn patients., Conclusion: We have achieved mixed hematopoietic chimerism-associated tolerance with high-dose HSCT, intrathymic donor renal tissue transplantation, and minimal conditioning without any adverse effects.

Introduction: We designed a prospective, randomized clinical trial to evaluate the immune response to thymic and peripheral infusions of donor haematopoietic stem cells (HSCs) to create tolerance in recipients of cadaver renal allografts., Method: We divided 24 patients into two equal groups. For group A, 350 ml of unfractionated bone marrow (BM) was aspirated from the anterior iliac crests of donor cadavers. A 2 ml aliquot of concentrated marrow was infused into the thymus of the subject and 100 ml into the BM before surgery; the remaining 250 ml was infused peripherally post-transplantation. The mean nucleated cell count inoculated into the thymus was 3.3 x 10(4) cells/cm(3) and into the periphery 8.6 x 10(7) cells/kg body weight. Group B (controls) underwent renal transplantation directly. Recipients were lymphocytotoxicity cross-match negative in both groups. Group A received low dose prednisolone and cyclosporin; controls also received azathioprine., Results: Over a mean follow-up of 703 days for both groups, group A had significantly better graft function with minimum acute rejection episodes or cytomegalovirus (CMV) infections, a mean serum creatinine (SCr) of 1.23 mg/dl and no graft or patient loss. Group B, with a mean SCr of 2.19 mg/dl had three patients with single acute rejection episodes, two of whom died following uncontrolled rejection-associated infections. The third patient maintained an SCr of 2.5 mg%. Actuarial graft survival was 87.5% in controls at the end of 2 years compared with group A with 100% graft survival at the end of 2 years., Conclusion: This novel approach of introducing unfractionated HSCs into the thymus and periphery to create tolerance is safe and efficacious and gives significantly better graft function, minimum acute rejection and no CMV disease with monotherapy.

We designed and implemented a clinical trial to achieve zero-rejection status in pediatric renal allograft recipients, using granulocyte-macrophage colony-stimulating factor (GM-CSF)-stimulated peripheral blood stem cell (PBSC) infusion. We studied 44 consecutive patients: 24 volunteers in a treated group (Tn) and 20 in a control group (Cn). Both groups were comparable with respect to clinical and laboratory parameters. The Tn group had 70.8% one haplo-match donors and the Cn group had 80% one haplo-match donors. Patients in the Tn group received cyclosporin A (CsA) and 0.4 mg/kg body weight prednisolone as immunosuppressants; azathioprine was added for patients of the Cn group, who received 1 mg/kg body weight prednisolone together with CsA. Living-related donors (LRD) of patients in the Tn group received GM-CSF 450 microg on four consecutive days followed by leucopheresis and immediate transfusion of unmodified PBSC into the recipient. This procedure was repeated once/twice, with one portal and one/two systemic infusions. Our aim was to maximize the dose of PBSC. The total average dose was 22 x 10(8) cells/kg body weight. Lymphocyte cross-match (LCM) was performed before GM-CSF injection and after the last PBSC infusion. Follow-up over an 18-month period revealed 100% graft survival with sustained low serum creatinine (SCr) values in patients of the Tn group as compared with 80% graft survival in patients of the control group who had marginally higher SCr levels. Absence of graft vs. host disease (GvHD), acute rejection episodes, and low incidence of cytomegalovirus (CMV) disease were the principal benefits of this protocol.